PROJECT SUMMARY CRISPR-Cas systems provide adaptive immunity in bacteria and archaea by employing guide RNAs and endonuclease effectors to specifically recognize and cleave invasive nucleic acids. The specific DNA targeting and cleavage activities of CRISPR-Cas systems have been adopted and developed for genome editing and various other applications, which are revolutionizing biomedical research and beyond. However, safety concerns are raised because of off-target genome editing and the dependence of these systems on endogenous host DNA repair pathways, hindering clinical application. Exploration of alternative CRISPR-Cas systems in nature not only offers an opportunity to overcome those challenges but may also inspire new applications. Structural and biochemical characterizations of CRISPR-Cas systems are critical for understanding their mechanisms and repurposing them for precise genome editing. Our long-term goals are to unravel the mechanisms underlying target nucleic acid recognition and cleavage mediated by type V and transposon-associated CRISPR-Cas systems, which provide essential knowledge for safer and more reliable application in treating human disease. In this proposal, we will work on the molecular mechanisms for four newly discovered CRISRP-Cas systems, covering DNA targeting (Cas12i), RNA targeting (Cas12g), and CRISPR RNA-guided DNA transposition (type I-F Cascade and Cas12k). As revealed in our preliminary data, Cas12i accommodates a longer crRNA-DNA heteroduplex than currently used Cas effectors, thus potentially improving specificity for genome editing. The RNA-guided RNase Cas12g is compact and thermostable, highlighting its potential for RNA editing and RNA targeting. Furthermore, type I-F Cascade and Cas12k direct transposition machinery for RNA-guided DNA transposition, opening a new paradigm for genome editing independent of DNA repair pathways. 1